Nucleic acids are highly functional macromolecules involved in accumulation and transmission of genetic information in living organisms. Specific recognition between nucleic acids or between a nucleic acid and a protein plays an important role in the function expression and regulatory function of the nucleic acid. With the recent rapid advances in genetic engineering and molecular biology, the mechanisms of such function expression have further been understood on a molecular level. A number of genes causative of diseases have been identified and the mechanisms of onset of such diseases have gradually been elucidated. With a background of such results, gene therapy has attracted attention by which therapy a disease is treated on a gene level using, as a therapeutic agent, a molecule capable of specific recognition in a manner mimicking the mode of recognition of a naturally occurring nucleic acid. The techniques of gene therapy which have so far investigated include, among others, the “gene transfer method” comprising artificially introducing a defective or deficient gene sequence into a patient's gene, the “antisense method” comprising binding an antisense molecule to mRNA in a base (nucleotide)-specific manner to thereby inhibit the synthesis of a protein causative of a disease, and the “antigene method” comprising binding an antisense molecule to a DNA region coding for a pathogenic protein to thereby inhibit the stage of transcription.
The so-called antisense molecule to be used in the “antisense method” or “antigene method” among the above-mentioned methods is required to have a number of characteristics, among which the following are important: 1) high-level ability to recognize a nucleic acid base sequence, 2) high stability in the form of complexes, 3) high stability against biological substances, in particular enzymes, 4) high cell membrane permeability, 5) ability to specifically interact with nucleic acids, and 6) nontoxicity to living bodies.
At first, attempts were made to use natural oligonucleotides as antisense molecules. Although these comparatively meet the above requirements 1) and 2), they have a problem in that they cannot meet the requirement 3) but are instantly decomposed enzymatically by means of nuclease occurring in vivo, so that the desired results cannot be attained.
The antisense molecules so far reported include, among others, 1) derivatives resulting from modification around the phosphate bonding, 2) derivatives resulting from modification of the glycosyl bonding or a hydroxyl group(s) in the ribose sugar portion, 3) derivatives resulting from modification of the base portion, and 4) nucleic acid model molecules having a skeletal structure other than the sugar-phosphate skeleton. More specifically, there may be mentioned such derivatives 1) as phosphorothioate type oligonucleotides resulting from substitution of a sulfur atom for an oxygen atom of phosphoryl group in the phospho-diester bonding and, further, phosphorodithioate type, phosphoroamidate type, methylphosphonate type and methylphosphonothioate type oligonucleotides; such derivatives 2) as α-anomer type oligonucleotides with the base coordinated in the sugar moiety in a manner reverse to the β-glucosyl bonding, oligonucleotides resulting from conversion of the sugar 3′-5′ phosphodiester bonding to the 2′-5′ bonding using a ribonucleotide, and 2′-methoxy derivatives resulting from methyl etherification at the 2′ position of ribose; such derivatives 3) as modified bases, for example 5-fluorouracil (5-FU) resulting from substitution of a fluorine atom at the 5 position of uracil, and fluorescent ethenoadenosine; such derivatives 4) as peptide nucleic acids (PNAs) having a peptide skeleton in place of the sugar-phosphate skeleton (O E. Uhlman, A. Peyman, Chem. Rev., 1990, 90, 544; O E. Uhlman, A, Peyman, G. Breipohl, D. W. Will, Angew. Chem. Int., Ed. Engl., 1998, 37, 2796, etc.).
The peptide nucleic acids (PNAs) so referred to herein are compounds which have attracted attention in view of such advantageous features as their base-specific recognizing ability, resistance to enzymolysis, very high affinity for nucleic acids owing to their having a neutral peptide chain as the main chain, and the possibility of arbitrary sequence being obtained in a simple and easy manner by the amide bond formation reaction. However, their excessively high affinity, for example their binding to targets, including mismatching, is rather disadvantageous, and drawbacks have also been reported, for example 15-mer or higher oligomers are nonspecifically adsorbed on intracellular proteins due to their having a hydrophobic peptide chain, or they are scarcely soluble in water.
Thus, there is room for improvement in antisense molecules, inclusive of PNAs.